Phytochemical investigation and anti-inflammatory potential of Atriplex leucoclada Boiss

Background The plant kingdom has long been considered a valuable source for therapeutic agents, however, some plant species still untapped and need to be phytochemically and biologically explored. Although several Atriplex species have been investigated in depth, A. leucoclada, a halophytic plant native to Saudi Arabian desert, remains to be explored for its phytochemical content and biological potentials. Herein, the current study investigated the metabolic content and the anti-inflammatory potential of A. leucoclada. Methods Powdered aerial parts of the plant were defatted with n-hexane then the defatted powder was extracted with 80% methanol. n-Hexane extract (ATH) was analyzed using GC–MS, while the defatted extract (ATD) was subjected to different chromatographic methods to isolate the major phytoconstituents. The structures of the purified compounds were elucidated using different spectroscopic methods including advanced NMR techniques. Anti-inflammatory activity of both extracts against COX-1 and COX-2 enzymes were examined in vitro. Molecular docking of the identified compounds into the active sites of COX-1 and COX-2 enzymes was conducted using pdb entries 6Y3C and 5IKV, respectively. Results Phytochemical investigation of ATD extract led to purification and identification of nine compounds. Interestingly, all the compounds, except for 20-hydroxy ecdysone (1), are reported for the first time from A. leucoclada, also luteolin (6) and pallidol (8) are isolated for the first time from genus Atriplex. Inhibitory activity of ATD and ATH extracts against COX-1 and COX-2 enzymes revealed concentration dependent activity of both fractions with IC50 41.22, 14.40 μg/ml for ATD and 16.74 and 5.96 μg/ml for ATH against COX-1 and COX-2, respectively. Both extracts displayed selectivity indices of 2.86 and 2.80, respectively as compared to 2.56 for Ibuprofen indicating a promising selectivity towards COX-2. Molecular docking study supported in vitro testing results, where purified metabolites showed binding affinity scores ranged from -9 to -6.4 and -8.5 to -6.6 kcal/mol for COX-1 and 2, respectively, in addition the binding energies of GC–MS detected compounds ranged from -8.9 to -5.5 and -8.3 to -5.1 kcal/mol for COX-1 and 2, respectively as compared to Ibuprofen (-6.9 and -7.5 kcal/mol, respectively), indicating high binding affinities of most of the compounds. Analysis of the binding orientations revealed variable binding patterns depending on the nature of the compounds. Our study suggested A. leucoclada as a generous source for anti-inflammatory agents. Supplementary Information The online version contains supplementary material available at 10.1186/s12906-023-04281-5.


Introduction
Inflammation is a self-defense mechanism that is triggered by pathogens, tissue injury, trauma or dysregulation of the normal physiological processes.Inflammatory response rises from the production of prostaglandins that is synthesized from the unsaturated arachidonic acid via cyclooxygenase (COX) enzymes [1,2].Cyclooxygenase enzymes are responsible for the formation of important biological mediators called prostanoids, including prostaglandins, prostacyclin and thromboxane.There are two known isoenzymes: COX-1 and COX-2.COX-1 is constitutively expressed in many tissues and is the predominant form in gastric mucosa and in kidney.COX-2 is not normally expressed in most cells, but elevated levels are found at sites of inflammation.Pharmacological inhibition of COX can provide relief signs of inflammation and pain [3].
Anti-inflammatory drugs include steroids and nonsteroid drugs.Steroid drugs have some serious side effects such as osteoporosis and fractures, immunosuppression, myopathy, cardiovascular disease, glaucoma and cataracts, diabetes and hyperglycemia, psychiatric disturbances, gastrointestinal and dermatologic adverse effects.These adverse effects limit their utility and make them less popular to be used in inflammatory diseases compared with nonsteroidal drugs [4].However, nonsteroidal drugs may have some side effects too such as bronchospasm, renal failure, thrombosis, and gastrointestinal bleeding [5].To overcome these problems, herbal medicines and phytochemicals have been submitted to studies to identify and develop natural products that can be used as anti-inflammatory agents [6][7][8] or as a combinatorial therapy with these synthetic drugs [9].
Atriplex leucoclada Boiss.(English name: cut-leaf saltbush, orach, Arabic name: Ragal, ‫,)رغل‬ is a low perennial shrub commonly growing in Saudi Arabian desert.This species has an agricultural importance in arid regions.It can adapt high salt habitats via different strategies [13,26].Reviewing the relevant literature little research was found discussing the metabolic content and/or the biological activity of A. leucoclada, where, one previous study reported the isolation of five triterpenoidal saponins and highlighted their molluscidal potential [27].Accordingly, this study was designed to add more research about the phytochemical constituents and antiinflammatory activity of A. leucoclada.

Plant material
Aerial parts of A. leucoclada were collected in October 2020 from the Qassim area, Kingdom of Saudi Arabia.The plant identity was verified by Ibrahim Aldakhil, area botanical expert, Qassim, KSA.Voucher sample number QPP-103 was deposited at the College of Pharmacy, Qassim University, KSA.

Preparation of extract
The dried aerial parts of A. leucoclada (700 g) were pulverized by a grinder and defatted with n-hexane (4 × 750 mL, at room temperature) to provide 1.7 g ATH extract.Afterwards, air-dried defatted powdered aerial parts were extracted with 80% methanol (4 × 1000 mL, at room temperature) to yield 45 g crude ATD extract.

In vitro determination of COX-1 and COX-2 enzymatic activity
COX-1 and COX-2 inhibition assays are based on the detection of the florescence produced by prostaglandin G2 (i.e., the intermediate product produced by the COX-1 and 2 enzymes).The assay was performed by using COX-1 inhibitor screening kit (#K548-100, BioVision Inc.) and COX-2 inhibitor screening kit (#K547-100, BioVision Inc.) to measure in vitro COX-1 and COX-2 enzymatic activities respectively.Ibuprofen was used as a positive control.Samples and control were used at different concentrations: 0.01-100 ug/ml.According to the manufacturer's instructions [2,30], 10 μL of samples or Ibuprofen was added to each well, and 80 μL of reaction master mix was prepared (76 μL of COX buffer assay, 1 μL COX probe, 2 μL diluted COX cofactor, 1 μL COX-1 or COX-2) and added to each well, and the fluorescence was measured kinetically at (Ex/Em = 535/587 nm) at 25°C for 5-10 min.The experiments were performed in triplicate.The relative percentage of inhibition of COX-1 and COX-2 was calculated according to the following Equation:

Statistical analysis
All measurements were performed in triplicate and results were expressed as mean ± standard deviation (SD).

In vitro determination of COX-1 and COX-2 inhibitory activity
In the present study, inhibitory effects of the defatted methanolic extract (ATD) and n-hexane (ATH) extract against COX-1 and COX-2 enzymes were examined in vitro.The results (Fig. 3A and B) showed that the tested extracts displayed inhibition of COX-1 and COX-2 enzymes in a concentration dependent manner being more selective towards COX-2 enzyme.Where IC 50 of ATD were 41.22, 14.4 μg/mL and of ATH were 16.74 and 5.96 μg/mL, against COX-1 and COX-2, respectively while ibuprofen IC 50 was 6.88 and 2.68, respectively (Table 2).

Discussion
Reviewing the relevant literature several studies were found reporting diverse chemical structures of the metabolic contents of some Atriplex species.While, in regards of A. leucoclada, previous investigations were not sufficient to describe the chemical profile of the plant.To achieve this purpose, the chemical composition of both defatted methanol (ATD) and hexane (ATH) extracts were investigated.The current findings showed that all purified compounds, except for compound (1), were isolated for the first time from A. leucoclada [46].Furthermore, compounds ( 6) and ( 8) were isolated for the first time from this genus.Compound (1) was previously obtained from other Atriplex species as A. inflata and A. nummularia [16,17], compounds (3) and (4) were previously in A. stocksii [15], compound (7) in A. canescens [47] and compound (9) in A. inflate [46], while compound (2) and ( 5) were identified by GC-MS only in the methanol extract of A. halimus [48,49].Interestingly, this study is the first to report GC-MS investigation of A. leucoclada.The current GC-MS analysis results enabled the tentative qualitative identification of numerous phytochemicals in ATH.The compounds identified have been interpreted as given in Table 1.Oxygenated and non-oxygenated hydrocarbons, alcohols, phenolics, steroidal and terpenoidal compounds were identified.Among the isolated compounds: phytol (21.24%) and cholest-5-en-3-ol (12.50%) are the major detected compounds.These results were similar to those reported for GC-MS analysis for A. lindleyi Moq [50].
Fig. 2 The most characteristic compounds identified in n-hexane (ATH) extract of A. leucoclada using GC-MS analysis Most of the compounds identified in the ATD or ATH extracts were previously reported to be more selective for COX-2.This is compatible with the current results that indicated high selectivity towards COX-2 enzyme.As selective COX-2, anti-inflammatory agents have minimum GIT side effects, and so they are more appreciated as per safety concern.Accordingly, the current findings acknowledge the metabolic content of A. leucoclada as a rich mixture of chemical entities that have promising potential of anti-inflammatory activity with limited side effects.
It is already stated that, computational studies played an effective role in drug development as it can provides a fast, cheap and easy method for expectation of the possible promising bioactive drugs.Herein, we examined the binding affinity of isolated (from ATD extract) as well as GC-MS identified compounds (in ATH extract) in this plant with key anti-inflammatory targets.Analysis of the binding with the two proteins COX-1 and COX-2 revealed variable binding patterns depending on the nature of the compounds.Steroids that constitute 44.44% of isolated compounds from ATD extract and 20.25% of detected compounds in ATH extract exhibited docking score with COX-2 ranged from -8.4 to -7.4 kcal/mol.During this study, we explored some data that can be gleaned from analysis of steroids conformation and interactions at the cyclooxygenase active site.Previous studies that examined the binding of the substrate "arachidonic acid" into COX active site suggested that the active site could be viewed as including three parts; proximal, central, and distal binding pockets and that the distal and proximal binding pockets are important for stabilization of the substrate, while the central part that contains the catalytic Tyr385 is the place where substrate is transformed into PGG 2 [69].Analysis of our docking results unveiled that steroids occupied the proximal binding pocket and may extends towards the central binding pocket.It was noted that presence of more hydrophilic groups at C-3 may contribute to the increased binding affinity as exemplified in β-sitosterol3-O-β-D-glucopyranoside (-8.4 kcal/ mol) with glucosylation at C-3 where it exhibited flat inverted conformation compared to that of β-sitosterol, furthermore, one of the sugar hydroxyl groups formed two hydrogen bonds with His90 and Gln192 in the side pocket, a pocket that was generated in COX-2 due to Ile523 mutation in COX-1 to the smaller Val523 in COX-2 [69] (Fig. 4B), while β-sitosterol (-7.4 kcal/ mol) exhibited only covalent bonds with Tyr355 at the Fig. 3 The in vitro inhibitory effect of n-hexane (ATH) and defatted methanol (ATD) extracts of A. leucoclada against COX-1 (A) and COX-2 (B) enzymes using Ibuprofen as a positive control.Data in the figures expressed mean ± SEM (n = 3).*** P < 0.001 consider statistically significant compared to ibuprofen.### P < 0.001 consider statistically significant compared to ATH group using one way ANOVA followed by Tukey's post hoc test constriction located near the entrance of the active site and with His90, Ser353, and His356 (Fig. 4A).Similarly, presence of carbonyl group at C-6 and hydroxyl groups at C-14, C-20, C-22, and C-25 as in the case of 20-hydroxyecdysone (-8.4 kcal/mol) may play a role in improving its binding affinity via formation of two hydrogen bonds between His356 and OH groups at C-20 and C-25, respectively and oxygen of the carbonyl group at C-6 exhibited two hydrogen bonds with Phe 580, and Ser581, in addition to hydrogen bonding between hydroxyl group at C-14 and Gln350 (Fig. 4C).Additionally, the current findings highlighted the flavonoid luteolin that was one of the lead metabolites exhibiting the highest binding affinity to COX-2 (-8.5 kcal/mol); analysis of the binding orientation of luteolin revealed that it formed one hydrogen bond with Trp387 in the catalytic region of the active site and another hydrogen bond with Asn382 in addition to π-π stacking with His388 and three π-alkyl interactions with Ala202, His207, and His386 (Fig. 4D).

Conclusion
The current study presented a detailed chromatographic exploration of A. leucoclada with successful isolation of nine compounds, eight of which were isolated for the first time from this species, moreover, GC-MS analysis identified the non-polar components of the hexane extract of the plant.These findings will add to chemical profile of this species, as well as to Atriplex genus.Additionally, COX-1 and COX-2 inhibitory activity testing noted the n-hexane and defatted methanol extracts for selective inhibitory activity against COX-2 enzyme.The molecular docking studies revealed high binding affinities and good binding interactions of most of the compounds with binding scores ranged from -8.5 to -6.6 kcal/mol for isolated compounds from ATD and -8.3 to -5.1 kcal/mol for compounds identified by GC-MS in ATH.Accordingly, A. leucoclada is suggested as a valuable source of safe anti-inflammatory agents.Future studies are recommended for evaluating the antiinflammatory potential of the highlighted metabolites whether, alone or in combination with commercially used anti-inflammatory drugs.

Table 1
Chemical profile of n-hexane extract (ATH) of A. leucoclada using GC-MS analysis

Table 3
Docking scores (kcal/mol) of isolated compounds from defatted methanolic (ATD) extract and compounds detected by GC-MS in n-hexane (ATH) extract of A. leucoclada against cyclooxygenase enzymes